Abstract Extracellular vesicles (EVs) are membranous particles released from nearly all cell types into all bodily fluids evaluated to date ? including serum and plasma. Depending on tissue of origin, health state and organism age, they carry a variety of complex cargo consisting of nucleic acids (5,000 microRNA documented to date), proteins (93,000 documented to date including cytokines) and metabolites. Due to their coordinate regulation of tissue homeostasis and biological processes through intercellular trafficking of microRNA and protein cargo, EVs are particularly attractive for this project because they can potentially serve as DIRECT biomarkers of aging, namely indicators AND mediators of the aging process. The goal of this project is to establish EVs with their microRNA and protein constituents as biomarkers of healthspan and lifespan and to inform biological mechanisms promoting healthspan and lifespan. We focus particularly on three of the hallmarks of aging, epigenetic alterations, cellular senescence and altered inter-cellular communication. Increasing evidence suggests that EVs secreted from senescent cells have unique characteristics and contribute to modulating the phenotype of recipient cells; thus, they have been newly deemed novel senescence associated molecular pattern (SASPs). We hypothesize expression of different amounts and different compositions of EVs are associated with different lifespan and healthspan of humans, and with different senescence states in murine models. In collaboration with Meso Scale Diagnostics, LLC (MSD), a premier developer of highly reliable and highly sensitive biomarker assays, we will develop new biomarkers of EVs informing aging mechanisms and test their function in vitro. These biomarkers will be qualified in the context of aging in our existing extensive human sample sets: individuals (n=3056) from multiple longitudinal cohort studies (EPESE aged >71 years; PALS aged 20-100 years) and NIH-funded controlled trials of geroprotective interventions (STRRIDE exercise aged 18-70 years; and CALERIE caloric restriction aged 22-45 years). Complementing this new biomarker development work, we will validate and qualify: the new S-PLEX high sensitivity (femtomole level detection) assays by MSD for soluble cytokines and circulating microRNAs we have identified as associated with healthspan and lifespan in elders. Taken together, we believe our broad expertise in biomarkers and aging, our interdisciplinary team and our partnership with a company with the capability to commercialize assays provide a unique project responsive to RFA-AG-18-018 for ?Development of valid reliable markers of aging-related biologic mechanisms for human studies?.